The present invention relates to the field of drug discovery and specifically, the design of prodrugs that are substrates for endogenous intracellular enzymes that are overexpressed in pathological cells.
Throughout and within this disclosure, various publications are referenced by first author and date, patent number or publication number. The full bibliographic citation for each reference can be found within the specification or at the end of this application, immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference into this disclosure to more fully describe the state of the art to which this invention pertains.
Cancer is one of the most commonly fatal human diseases worldwide. Treatment with anticancer drugs is an option of steadily increasing importance, especially for systemic malignancies or for metastatic cancers that have passed the state of surgical curability. Unfortunately, the subset of human cancer types that are amenable to curative treatment today is still rather small (Haskell, C. M. (1995)) resulting in about 600,000 deaths per year. See, Cancer Facts and Figures, 1999 American Cancer Society. Progress in the development of drugs that can cure human cancer is slow, with success limited to a few hematological malignancies and fewer solid tumor types (Dorr and Van Hoff (1994)). Progress in discovering therapies that are based upon disease mechanism offers opportunities for future success. (Cobleigh, M. A. et al. (1999); and Roth, J. A. et al. (1999)).
The heterogeneity of malignant tumors with respect to their genetics, biology and biochemistry as well as primary or treatment-induced resistance to therapy mitigate against curative treatment. Moreover, many anticancer drugs display only a low degree of selectivity, causing often severe or even life threatening toxic side effects, thus preventing the application of doses high enough to kill all cancer cells. Searching for anti-neoplastic agents with improved selectivity to treatment-resistant pathological, malignant cells remains therefore a central task for drug development.
Cancer cells are characterized by uncontrolled growth, de-differentiation and genetic instability. The instability expresses itself as aberrant chromosome number, chromosome deletions, rearrangements, loss or duplication beyond the normal diploid number. (Wilson, J. D. et al. (1991)). This genomic instability may be caused by several factors. One of the best characterized is the enhanced genomic plasticity which occurs upon loss of tumor suppressor gene function (e.g., Almasan, A. et al. (1995a) and Almasan, A. et al. (1995b)). The genomic plasticity lends itself to adaptability of tumor cells to their changing environment, and may allow for the more frequent mutation, amplification of genes, and the formation of extrachromosomal elements (Smith, K. A. et al. (1995) and Wilson, J. D. et al. (1991)). These characteristics provide for mechanisms resulting in more aggressive malignancy because it allows the tumors to rapidly develop resistance to natural host defense mechanisms, biologic therapies (see, Wilson, J. D. et al. (1991) and Shepard, H. M. et al. (1988)), as well as to chemotherapeutics (see, Almasan, A. et al. (1995a); and Almasan, A. et al. (1995b)).
In addition, the clinical usefulness of a chemotherapeutic agent may be severely limited by the emergence of malignant cells resistant to that drug. A number of cellular mechanisms are probably involved in drug resistance, e.g., altered metabolism of the drugs, impermeability of the cell to the active compound or accelerated drug elimination from the cell, altered specificity of an inhibited enzyme, increased production of a target molecule, increased repair of cytotoxic lesions, or the bypassing of an inhibited reaction by alternative biochemical pathways. In some cases, resistance to one drug may confer resistance to other, biochemically distinct drugs. An alternative mechanism of resistance to cancer chemotherapeutics occurs via the functional loss of tumor suppressor genes. The best characterized of these are p53, RB and p16. Funk, J. O. 1999; and Teh, B. T. (1999)). Loss of function of these gene products leads to depressed expression of enzymes commonly targeted by anti-cancer drugs (e.g., 5-fluorouridyl (xe2x80x9c5FUxe2x80x9d)/thymidylate synthase and methotrexate/dihydrofolate reductase). (Lee, V. et al. (1997); Lenz, H. J. et al. (1998); and Fan, J. and Bertino, J. (1987)). Amplification of certain genes is involved in resistance to biologic and chemotherapy. Amplification of the gene encoding dihydrofolate reductase is related to resistance to methotrexate, while overexpression/amplification of the gene encoding thymidylate synthase is related to resistance to treatment with 5-fluoropyrimidines. (Smith, K. A. et al. (1995)). Table 1 summarizes some prominent enzymes in resistance to biologic and chemotherapy.
The poor selectivity of anticancer agents has been recognized for a long time and attempts to improve selectivity and allow greater doses to be administered have been numerous. One approach has been the development of prodrugs. Prodrugs are compounds that are toxicologically benign but which may be converted in vivo to therapeutically active products. In some cases, the activation occurs through the action of a non-endogenous enzyme delivered to the target cell by antibody (xe2x80x9cADEPTxe2x80x9d or antibody-dependent enzyme prodrug therapy (U.S. Pat. No. 4,975,278)) or gene targeting (xe2x80x9cGDEPTxe2x80x9d or gene dependent enzyme-prodrug therapy (Melton, R. G. and Sherwood, R. E. (1996)). These technologies have severe limitations with respect to their ability to exit the blood and penetrate tumors. See, Connors, T. A. and Knox, R. J. (1995).
A number of nucleotide and nucleoside analogs have been developed and tested as both anti-tumor and anti-viral agents. For example 5-flurouracil (5FU) and 5-fluorodeoxyuridine (5FUdR) have been widely used as chemotherapeutic agents based on their ability to be converted intracellularly into inhibitors of the thymidylate synthase (TS) enzyme. As with many other enzyme inhibitory chemotherapeutic agents, cancer therapy using 5FU frequently leads to selection for aggressive drug resistant tumor cells which are refractory to further treatment with this drug. (Aschele, C. et al. (1994); Mader, R. M. et al. (1998); Lxc3x6nn, U. et al. (1996); Paradiso, A. et al. (2000); and Edler, D. et al. (2000)).
Particular attention has also been paid to halogenated nucleoside analogs such as (E)-5-(2-Bromovinyl)-2xe2x80x2-deoxyuridine (BVdU). These types of compounds were originally developed as anti-viral agents based on the observation that they required phosphorylation to the monophosphate nucleotide form in order to elicit cytotoxic responses and this phosphorylation was preferentially accomplished by herpes virus thymidine kinase (TK) enzymes. BVdU and related compounds have consistently shown limited toxicity to normal mammalian cells, while they have been effective at killing virally infected cells. (DeClerq, E. et al. (1997)).
Throughout the extensive testing of nucleoside analogs such as BVDU as anti-viral and anti-cancer agents, they have consistently been reported to have minimal toxicity to both normal and cancer cells. These observations have been supported by results demonstrating the preferential phosphorylation of such compounds by viral thymidine kinase enzymes, which explains the low toxicity of these compounds for mammalian cells. Thus, BVdU and related nucleoside analogs have not been developed as anti-cancer therapeutic agents. (De Clerq, E. et al. (1997)).
This invention provides novel compounds that selectively inhibit the proliferation of pathological cells, for example, pathological cells that endogenously overexpress a target enzyme that confers resistance to biologic and chemotherapeutic agents. The enzyme acts on a prodrug compound of this invention to 1) convert it to a cellular toxin and/or 2) release a toxic byproduct. In another aspect of this invention, the product of an initial reaction with a resistance enzyme, is subsequently fully activated by a common cellular enzyme such as an acylase, phosphatase or other xe2x80x9chousekeepingxe2x80x9d enzyme (Voet, et al. (1995)) or common cellular constituent (e.g., water) to release the toxic byproduct from the prodrug.
In one embodiment, the activity of the target enzyme has been greatly enhanced in a target cell as a result of loss of tumor suppressor function and/or selection resulting from previous exposure to chemotherapy. In another embodiment, the pathological cell contains a target enzyme that is an expression product of an infectious agent in the cell. The expression product can confer antibiotic resistance.
Another aspect of this invention includes assays for screening for new prodrugs that can be activated by target enzymes. Kits to perform such assays containing the reagents and instructions necessary to complete the assay and analyze the results are also is provided by this invention.
Further provided by this invention is a method for treating a subject by delivering to the subject a prodrug as described herein. The prodrugs of this invention may be used alone or in combination with other chemotherapeutics or alternative anti-cancer therapies such as radiation.
A further aspect of this invention is the preparation of a medicament for use in treating a subject suffering from a pathology characterized by cells expressing a target enzyme.
A still further aspect of this invention is a method for identifying the optimal therapeutic for a subject, by isolating cells expressing a target enzyme and contacting the cells with at least one of the prodrugs of this invention, and then identifying which of the one or more prodrugs inhibits the proliferation or kills the cells, thereby identifying the optimal therapeutic for the subject.
The prodrugs of this invention have the following structure: 
or tautomers thereof;
wherein R12 or R13 may be the same or different and are selected from the group consisting of oxo, OH or NHNH2;
wherein if a is 0 or 1, providing that if a is 0 and R13 is oxo, then a double bond exits between position 3 and 4 and R12 is NHNH2 or
if a is 0 and R12 is oxo, then a double bond exists between position 2 and 3 and R13 is NHNH2; or
if a is 1, then R12 and R13 are both oxo;
wherein R1 is a substituent having the formula: 
wherein R2 and R3 are independently an unsaturated or a saturated hydrocarbyl group;
wherein n is 0 or an integer from 1 to 10 and m is 0 or 1; and
wherein R4 is selected from the group consisting of F, Cl, Br, I, CN, SO3H, CO2H, CO2CH2CH3, CO2CH3, SI(CH3)3, CHO, NO2, CF3, CCl3,
CHxe2x95x90C(R15)2 or a substituent having the structure: 
wherein Xa and Xb are independently the same or different and are selected from the group consisting of Cl, Br, I, and a potent leaving group;
wherein Ya, Yb or Yc are independently the same or different and are H or F;
wherein Z, Za and Zb are independently the same or different and are O or S; and
wherein R14 is H or F, providing if R14 is F, then a is 1 and R12 and R13 are both oxo; and
wherein Q is selected from the group consisting of a sugar, a carbocyclic, and an acyclic compound, or a masked phosphate derivative or phosphoramidate derivative thereof.